The transformation of living cells by bombardment with biological substances such as heterologous DNA or RNA is described in a patent application entitled Method For Transporting Substances Into Living Cells and Tissues and Apparatus Therefor, by Sanford et al., U.S. Ser. No. 06/670,771, filed Nov. 13, 1984 now U.S. Pat. No. 4,945,050. An improved apparatus for delivering substances into cells and tissue is described in a patent application filed by Sanford et al. entitled "Biolistic Apparatus for Delivering Substances into Cells and Tissues in a Non-Lethal Manner," U.S. Ser. No. 07/161,807 filed Feb. 29, 1988, also by Sanford et al. The process described involves accelerating particles of an appropriate size to high velocities sufficient to penetrate cell walls and/or cell membranes thereby entering into the cell's cytoplasm, nucleus, or organelles. If the particles are carrying biological substances and the velocity is sufficient to penetrate the cell wall without destroying the cell, the biological substances are introduced into the cell.
In this process, holes formed in the cell membrane need not be any larger than would be achieved using microinjection procedures and need only remain open for a fraction of a second. Biological substances such as RNA and DNA may be carried into the cells by a variety of mechanisms such as precipitating the DNA onto the surface of inert particles such as tungsten spheres, latex beads or ferrite crystals as is described by Sanford et al. DNA in a liquid carrier may be used as well or DNA in solid form may constitute the particles themselves. This process is particularly advantageous because large numbers of cells can be bombarded simultaneously and does not require the manipulation of individual cells.
This process is also described by Sanford et al. in J. Part Sci. and Tech. 5:27-37 (1987) and is summarized in a recent review by Sanford in Trends in Biotechnology 6:229-302 (1988). The biolistic delivery of DNA into onion cells is described by Klein et al. in Nature 327:70-73 (1987). The biolistic delivery into corn cells is described by Klein et al. in Proc. Natl. Acad. Sci. 85:4305-4309 (1988) and Biotechnology 5:559-563 (1988). The transformations of microbes and organelles was first shown by Johnston et al. in Science 240:1538-1541 (1988) and Boynton et al. in Science 240:1534-1538 (1988) and Armeleo in Current Genetics 17:97-103 (1990). Organelle transformation was further elucidated by Fox et al. in PNAS 85:7288-7292 (1988), Blowers et al. The Plant Cell 1:123-132 (1989) and Daniell et al. PNAS (in press) (1989).
The utility of the process for higher plant transformation has been reviewed by Sanford Physiologia Plantarum (in press) (1989) and has been further demonstrated in numerous papers. Further the transformation of higher animal cells using this bombardment technique has been demonstrated in vitro in Zelenin et al. FEBS Letters 244: 65-67 (1989) Johnston et al. (in preparation) and Williams et al. Nature (submitted) (1990) and most recently in vivo in Johnston et al. (in preparation) Zelenin et al. (in preparation).
Based on these early teachings, various apparatus have been constructed to effect the delivery of particles carrying biological substances.
The apparatus described by Sanford et al. is commercially available from E. I. du Pont de Nemours and Company, Wilmington, Del. in the form of a gunpowder driven device in which the hot gasses generated by a gunpowder discharge form a hot gas shock wave which accelerate a macroprojectile carrying many tiny microprojectiles. When the macroprojectile strikes a stopping plate having a hole therein, the microprojectiles continue their travel, striking the target cells. Morikawa et al. Appl. Microbiol. Biotechnol. 31:320-322 (1989) have constructed a device based on the teachings of the Sanford et al. that uses pressurized nitrogen to drive the macroprojectile. Agracetus in European patent application 8731062.4 filed Dec. 2, 1987 (publication number 0270356) describe a macroprojectile accelerator. When the macroprojectile is a thin disk, it is accelerated by the use of a very high voltage discharge, which vaporizes a droplet of water, creating a hot gas which drives the projectile similar to the hot gases generated by the gunpowder discharge.
Dr. Laurie Mets has built a particle accelerator which is based on the same principle described in the Sanford et al. patent application, namely, gas flow entrainment of the particles. Finally Oard et al. (1990) achieved transient gene expression in maize, rice and wheat cells using compressed air to accelerate a cylindrical macroprojectile. Plant Physiology 92:334-339 describes the use of such an airgun for propelling DNA coated microprojectiles. A polycarbonate vacuum chamber encloses the airgun muzzle and target material.
While these various apparatus represent various approaches to the problem of how best to accelerate microprojectiles for transferring biological materials into cells and tissues, they all suffer from one or more deficiencies. First of all, many of the apparatus constructed are not flexible as to mode of use. There are a wide array of applications for the ballistic process, each calling for different capabilities, settings, configurations, etc. The existing apparatus tend to be optimal for a single mode of use or for a single application but are not adaptable to plural needs. Secondly, the apparatus available generally does not provide the degree of repeatable results, target-to-target and day-by-day, that is desired. Existing accelerators have a high degree of variability in performance. Also the particle dispersion pattern is poor and not uniform over the target area. Existing apparatus often does not effectively break up particle aggregates and does not allow control over how large an area over which the particles are dispersed.
Most of the existing apparatus is bulky, generally immobile and generally cannot be handheld such as is needed for some veterinary or medical applications. In this connection, most of the apparatus that are available requires that the targets be placed inside a vacuum chamber and are not suited for use on targets larger than such chamber. Whatever the target used, there is a tendency for the accelerator apparatus to damage (or kill) a certain number of the cells, which impairs cell division or cell differentiation. This is especially true when the distance to the target needs to be short, as in medical applications. It is desirable therefor to have better velocity control, less gas blast, less acoustic shock, less high velocity debris, less heat, and less radiant energy.
Apparatus which employs gunpowder or high voltage discharges tend to generate high temperatures. Furthermore, high voltage discharges generate a blinding flash which may generate ultraviolet light and other forms of ionizing radiation. Such may be harmful to the cells being transformed, or the DNA being delivered.